Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters’ positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.

Finally, we added a repression mechanism.The repressors (Rep) binding rate to a free promoter is k r and the unbinding rate is k a (reactions S5 and S6).When Rep is bound, the promoter is unavailable to RNAP binding.The influence of repressors can be strongly reduced by inducers, Ind.These can directly bind/unbind repressors (at rates k ind / k unind ) (reaction S7 and S8).When bound by an inducer, the repressor cannot bind to the promoter.To model non-overlapping tandem promoters, we adapted a model for operons with multiple promoters in tandem formation, recently published in 6 .First, we identify the promoters according to their position (Pro U and Pro D , where U and D stand for upstream and downstream, respectively).Similarly, we identify elongating RNAPs (RNAP e ) and RNAs, depending on which promoter they start from.Given this, Reactions S9 and S10 model transcription initiation of the downstream and upstream promoter, respectively, as follows: Then, the following events can occur to RNAP e U .First, if the downstream promoter is unoccupied, RNAP e U can pass by and complete the RNA (reaction S14):

𝑃𝑟𝑜 + 𝑅𝑁𝐴𝑃
Contrarily, if the downstream promoter is occupied by an RNAP, RNAP e U will collide with it.In that case: (i) either RNAP e U will fall-off (reaction S15); or (ii) both RNAPs fall-off (reaction S16): S15 and S16 are competing reactions, and one of them has to occur.We control their mean rate using a base rate constant (k c ).Meanwhile, we control their relative frequency by multiply k c with either f 1 or f 2 , respectively, where: Here, f 1 stands for the frequency with which only RNAP e U falls-off, while f 2 stands for the frequency with which both RNAPs fall-off.
Noteworthy, at quasi-equilibrium, the RNAP collisions will affect mean RNA levels, only when causing RNAP fall-offs.Thus, since we are focusing on mean RNA levels, we do not model collisions that do not cause fall-offs.Also, we expect little differences in mean RNA levels at quasi-equilibrium between when the RNAP that falls-off is RNAP e U or, instead, is the RNAP at the downstream promoter.Thus, we only model the first case (since the binding of RNAP e U to DNA should be comparatively weaker 7 ).
Finally, we modeled external repression and activation of the promoters.Reaction S17-S18 and S19-S20 model the binding of specific repressors, Rep U and Rep D , to the upstream and downstream promoters, respectively: Finally, we considered that, if the downstream promoter subject to repressors, the RNAP elongating from the upstream promoter could collide with a bound repressor.When occuring, similarly to when the downstream promoter is occupied by an RNAP, either RNAP e U will fall-off (reaction S25, with frequency f r ) or the bound repressor will fall-off (reaction S26, with frequency (1-f r )).For simplicitly, we do not model cases where both repressor and RNAP fall-off since, given the number of repressors and their binding and unbinding rate constants, this simplification did not influence the dynamics.
+   .    •  →   .  +  (S25) The RNAs produced in reactions S12 and S14 can decay via reactions S27 and S28, respectively: Finally, all parameter values and supporting references are shown in Table S1.Data for when uninduced, when fully induced (all promoters), and when fully induced while also subject to rifampicin (2.5 µg/mL and 5.0 µg/mL).The shaded areas represent the SEM from 3 biological replicates."Induced" stands for when all promoters of the construct are fully induced."Uninduced" stands for when no promoter is induced.The insets show the same data when zooming a smaller range of y values.S3.The dash line is the line of equality (E D ind = E T ).Finally, the insets show zoomed areas of the plots, to better distinguish which data points are above and below the dashed line.

Figure S5:
In silico estimation of α R for non-overlapping tandem promoters when (A) the upstream promoter is gradually induced, and, when (B) the downstream promoter is gradually induced.The plots are in logarithmic scale.In (A) the downstream promoter is fully induced while in (B) the upstream promoter is fully induced.f r stands for the relative frequency with which an RNAP from the upstream promoter falls-off upon colliding with a repressor bound to the downstream promoter (as opposed to the repressor falling-off).For f r = 1, it is always the RNAP that falls-off with the collision.

Figure S3 :
Figure S3: Average single-cell fluorescence levels of individual and non-overlapping tandem promoters.We measured population fluorescence levels (by spectrophotometry) and normalized them by the corresponding O.D. 600 to get a proxy for the average single-cell fluorescence levels (Methods section "Spectrophotometry").Data for when uninduced, when fully induced (all promoters), and when fully induced while also subject to rifampicin (2.5 µg/mL and 5.0 µg/mL).The shaded areas represent the SEM from 3 biological replicates."Induced" stands for when all promoters of the construct are fully induced."Uninduced" stands for when no promoter is induced.The insets show the same data when zooming a smaller range of y values.

Figure S4 :
Figure S4: In silico expression levels of individual downstream promoters (E D ind ) and non-overlapping tandem promoters (E T ).(A) Effects of changing the RNAP escape rate from the downstream promoter (k esc D ).Each colored line represents a different relative frequency (f 2 ) of both RNAPs falling-off upon colliding (as opposed to only one RNAP falling-off).(B) Effects of changing both the rates of RNAP binding and of RNAP escape from the upstream and from downstream promoter (k t U , k esc U , k t D , and k esc D ).Each colored line represents a different set of parameter values modeling different conditions.Each condition ("Cond") is described in TableS3.The dash

Figure S6 :
Figure S6: Induction curves of P LacO3O1 (inducible by IPTG), P tetA (inducible by aTc), and P BAD (inducible by arabinose) in individual formation, as measured by single-cell fluorescence.The mean and standard error of the single-cell fluorescence were extracted from 3 biological replicates in each condition.In all cases, the strongest inducer concentration resulted in a minor decrease in fluorescence.For this reason, the previous condition was used as being "maximum induction".

Figure S7 :
Figure S7: Fluorescence levels of DH5α-Pro cells (WT) absent of our plasmids when subject to rifampicin.Population fluorescence levels (spectrophotometry) are normalized by the corresponding O.D. 600 , to be used as a proxy for average single-cell fluorescence (Methods section "Spectrophotometry").We measured 180 minutes after adding rifampicin, when reaching O.D. 600 of 0.3.The error bars represent the SEM from 3 biological replicates.

Figure S8 :
Figure S8: Distribution of inter-promoter spacing between TSSs of non-overlapping tandem promoters (in bp).The red dotted line marks the 350 bp spacing.Approximately 85% of promoters in tandem formation are spaced by less than 350 bp.

Figure S9 :
Figure S9: Mean single-cell expression levels (E) of synthetic tandem constructs differing only in inter-promoter spacing between non-overlapping tandem TSSs.The (very small) black error bars represent the standard error of the mean while the red error bars represent the standard deviation of the 3 technical replicates.

Modeling non-overlapping tandem promoters with transcriptional interference due to RNAP collisions leading to fall-offs with repression and induction mechanisms.
These repressors can be inactivated by the binding of their respective inducers, Ind U and Ind D , respectively (reaction S21-S22 for Rep U and reaction S23-S24 for Rep D ), as follows: